EP2711417B1 - Utilisation de polymères hydrophobes poreux revêtus et remplis de liquides hydrophobes immiscibles avec l'eau pour surfaces répulsives de protéines et cellules - Google Patents

Utilisation de polymères hydrophobes poreux revêtus et remplis de liquides hydrophobes immiscibles avec l'eau pour surfaces répulsives de protéines et cellules Download PDF

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EP2711417B1
EP2711417B1 EP12006593.3A EP12006593A EP2711417B1 EP 2711417 B1 EP2711417 B1 EP 2711417B1 EP 12006593 A EP12006593 A EP 12006593A EP 2711417 B1 EP2711417 B1 EP 2711417B1
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Prior art keywords
hydrophobic
areas
porous
polymer layer
water
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German (de)
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EP2711417A1 (fr
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Erica Boles
Pavel Dr. Levkin
Ursula Prof. Dr. Obst
Thomas Dr. Schwartz
Junsheng Li
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Karlsruher Institut fuer Technologie KIT
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Karlsruher Institut fuer Technologie KIT
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Priority to US14/031,912 priority patent/US20140087173A1/en
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B3/00—Layered products comprising a layer with external or internal discontinuities or unevennesses, or a layer of non-planar form; Layered products having particular features of form
    • B32B3/26—Layered products comprising a layer with external or internal discontinuities or unevennesses, or a layer of non-planar form; Layered products having particular features of form characterised by a particular shape of the outline of the cross-section of a continuous layer; characterised by a layer with cavities or internal voids ; characterised by an apertured layer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062—General methods for three-dimensional culture
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
    • B05D—PROCESSES FOR APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
    • B05D5/00—Processes for applying liquids or other fluent materials to surfaces to obtain special surface effects, finishes or structures
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M39/00—Means for cleaning the apparatus or avoiding unwanted deposits of microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2535/00—Supports or coatings for cell culture characterised by topography
    • C12N2535/10—Patterned coating
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00—Stock material or miscellaneous articles
    • Y10T428/249921—Web or sheet containing structurally defined element or component
    • Y10T428/249953—Composite having voids in a component [e.g., porous, cellular, etc.]

Definitions

  • the present invention relates to a substrate with a solid support coated with a porous hydrophobic polymer layer or film, wherein said polymer layer or film is coated with a water-immiscible hydrophobic liquid forming a stable film on the polymer surface.
  • the present invention further relates to a method of forming a protein- and cell-repellent surface, comprising the steps of providing a substrate with a solid support coated with a porous hydrophobic polymer layer or film, and applying a water-immiscible hydrophobic liquid to said porous hydrophobic polymer layer or film, a respective method of preventing the formation of biofilms, adhesion of cells and bacteria, and/or migration of cells and bacteria on a surface, as well as the use of a porous hydrophobic polymer layer or film that is coated with a water-immiscible hydrophobic liquid for forming a protein-, cell- and bacteria-repellent surface.
  • Protein-, cell- and bacteria-repellent surfaces are of relevance in a wide range of applications including protein, cell, and DNA microarrays, cell patterning, microfluidic devices, biological implants, and non-biofouling surfaces.
  • cell adhesion e.g. of bacterial cells
  • cell adhesion and biofilm formation represent a complex and serious problem for industry and biomedicine. Over 60% of microbial infections in humans are caused by biofilm-forming bacteria. Biofilms also can cause very serious problems in industrial water systems.
  • biofilms can protect their constituent cells in various ways and some cells in biofilms exist in a slow-growing and/or starved state, or adopt a distinct and protected phenotype. Moreover, biocides often cannot penetrate the full depth of the biofilm. The development of a protein- and cell-repellent surface would help to prevent or retard biofilm formation.
  • cell microarrays require thousands of spatially separated spots of the effectors, such as proteins, small molecules, oligonucleotides, or transfection reagents, so that they only affect the cluster of cells growing on top of each spot and a clear read-out can be obtained. Consequently, large spot-to-spot distances are usually required to separate each spot.
  • the primary limitation of current cell microarrays is that although the effectors are spatially separated, usually the whole surface is seeded with cells and thus the affected and non-affected cells are not physically separated. This also makes it difficult to discern the affected cells within the background of non-affected cells for microscopic analysis.
  • creating cell microarrays that spatially separate, but also confine, both the effector and the affected cell cluster from one spot to another will significantly ease conducting e.g. high-throughput screens.
  • Non-biofouling surfaces to limit protein and cell adhesion.
  • Polyethylene glycol (PEG) is most commonly used for this purpose, but other coatings such as pluronics F127 (block copolymers based on ethylene oxide and propylene oxide) and hyaluronic acid (HA) are also used.
  • pluronics F127 block copolymers based on ethylene oxide and propylene oxide
  • HA hyaluronic acid
  • Many different surface chemistry techniques to prevent protein and cell adhesion have been reported.
  • many of the surface chemistries used to create non-biofouling surfaces are limited in their method of immobilizing the molecules on the surface. Simply immersing the surface in a chemical solution or stamping the molecule on the surface can result in a non-uniform, low-density, and unstable coating.
  • Micro-contact printing and dip pen nanolithography are also used to print non-biofouling molecules on surfaces, but they require expensive equipment.
  • There are methods to covalently link molecules to surfaces such as modified alkanethiolate self-assembled monolayers (SAMs), but these modified SAMs can still adsorb significant amounts of serum proteins and they tend to oxidize under ambient conditions, thus restricting their use to short-term experiments.
  • SAMs modified alkanethiolate self-assembled monolayers
  • cell patterning techniques require serum-free or serum-depleted cell seeding conditions, in addition to surface modification, to limit protein and cell adhesion in the desired areas. This can affect the condition of the cells and possibly the experimental results.
  • aqueous two-phase systems have been introduced for cell patterning, but improvements in the spatial control and high-throughput possibilities are still needed.
  • biocide-releasing surfaces have long been used to kill bacteria. However, such biocide-releasing surfaces get exhausted over time.
  • HEMA-EDMA hydroxyethyl methacrylate-co-ethylene dimethacrylate
  • PFPMA 2,2,3,3,3-pentafluoropropyl methacrylate
  • WO 2012/100099 A2 describes self-healing, slippery liquid-infused porous surfaces (SLIPS), as well as methods of making and using the same.
  • Epstein et al. Epstein, A. K. et al.; PNAS, 109(33); 2012; pp. 13182-13187
  • EP 2 481 794 A1 describes a method for making arrays or patterns comprising hydrophilic areas and hydrophobic background, as well as related methods of cultivating cells thereon.
  • Li et al. Li, J. S. et al.; Langmuir, 28; 2012; pp.
  • the technical problem underlying the present invention is to provide an improved protein-, cell- and bacteria-repellent substrate, as well as a simple and rapid method of forming an improved stable protein-, cell- and bacteria-repellent surface, as well as an improved method of preventing the formation of biofilms, adhesion of cells and bacteria and/or migration of cells and bacteria on a surface.
  • the present invention relates to a substrate with a solid support coated with a porous hydrophobic polymer layer or film, wherein said polymer layer or film is coated with a water-immiscible hydrophobic liquid forming a stable film on the polymer surface.
  • the substrate, the solid support, and the porous hydrophobic polymer layer or film are as defined hereinafter.
  • the porous hydrophobic polymer layer or film comprises a crosslinked polyvinyl monomer and a monovinyl monomer that have been copolymerized in the presence of an inert porogen.
  • Said polyvinyl monomer, monovinyl monomer, and inert porogen can be as defined hereinafter.
  • the water-immiscible hydrophobic liquid of the substrate of the present invention is selected from the group consisting of perfluorinated lubricants, perfluorinated hydrocarbons, and organosilicone compounds. More preferably, said liquid is a perfluorinated lubricant, selected from the group consisting of perfluoropolyethers, tertiary perfluoroalkylamines, perfluorotri-n-butylamine, perfluoroalkylsulfides, perfluoroalkylsulfoxides, perfluoroalkylethers, perfluorocycloethers, perfluoroalkylphosphines, perfluoroalkylphosphineoxides, long-chain perfluorinated carboxylic acids, fluorinated phosphonic acids, fluorinated silanes, and combinations thereof.
  • perfluorinated lubricant selected from the group consisting of perfluoropolyethers, tertiary
  • said liquid is a perfluoropolyether, selected from the group consisting of fluorine end-capped hexafluoropropylene epoxide homopolymers, and the KRYTOX ® family of lubricants by DuPont ® .
  • porous hydrophobic polymer layer or film not the entire above porous hydrophobic polymer layer or film is hydrophobic, but the porous hydrophobic polymer layer or film comprises
  • hydrophilic areas and the hydrophobic areas can be as defined hereinafter.
  • the present invention relates to a method of forming a protein-, cell- and bacteria-repellent surface, comprising the steps of:
  • protein-, cell- and bacteria-repellent describes the fact that the surfaces formed in the method of the present invention do not allow or do constrain the attachment of any proteins, cells or bacteria. Accordingly, said surfaces do not allow or do constrain the attachment of viruses, prokaryotic cells such as bacteria, eukaryotic cells, or multicellular organisms such as plants or sessile marine organisms.
  • Means for applying a water-immiscible hydrophobic liquid to said porous hydrophobic polymer layer or film are not particularly limited and include for example the application of said liquid with a syringe or a pipette to the substrate and subsequent washing of the substrate, e.g. with water.
  • porous hydrophobic polymer layer or film not the entire above porous hydrophobic polymer layer or film is hydrophobic, but the porous hydrophobic polymer layer or film comprises
  • the surface that is formed in the method of the present invention is a flat surface and does not contain any physical barriers, such as wells, recesses, indentations, depressions, ridges or the like.
  • the water-immiscible hydrophobic liquid, the substrate, the solid support, the porous hydrophobic polymer layer or film, and the hydrophilic and hydrophobic areas are as defined above or hereinafter.
  • the present invention relates to a method of preventing or retarding the formation of biofilms, adhesion of cells and bacteria, and/or migration of cells and bacteria on a surface or on parts of a surface, comprising the steps of:
  • biofilm as used herein relates to any aggregate of cells in which the cells adhere to each other on a surface.
  • Cells that form a biofilm may be prokaryotic or eukaryotic cells or viruses.
  • the surface on which prevention or retardation of the formation of biofilms is intended is not particularly limited, provided that a porous hydrophobic polymer layer or film in accordance with the present invention can be coated on said surface.
  • the water-immiscible hydrophobic liquid, means for applying the same to said porous hydrophobic polymer layer or film, as well as said porous hydrophobic polymer layer or film are as defined above or hereinafter.
  • the present invention relates to the use of a porous hydrophobic polymer layer or film that is coated with a water-immiscible hydrophobic liquid for forming a protein-, cell- and bacteria-repellent surface.
  • the water-immiscible hydrophobic liquid, the surface, means for applying the same to said porous hydrophobic polymer layer or film, as well as said porous hydrophobic polymer layer or film, are as defined above or hereinafter.
  • cell array refers to a substrate comprising a multitude of separated and confined areas wherein cells can be kept in an aqueous medium. According to the present invention, said cells cannot grow on or migrate across the hydrophobic areas being coated with a water-immiscible hydrophobic liquid.
  • cell as used in this context is not particularly limited and relates to any prokaryotic or eukaryotic cells.
  • the substrate, the solid support, the porous hydrophobic polymer layer or film, the hydrophilic and hydrophobic areas, and the water-immiscible hydrophobic liquid are as defined above or hereinafter.
  • porous hydrophobic polymer layer or film that is coated with a water-immiscible hydrophobic liquid for preventing the migration of cells across said porous hydrophobic polymer layer or film.
  • the substrate, the solid support, the porous hydrophobic polymer layer or film, the hydrophilic and hydrophobic areas, and the water-immiscible hydrophobic liquid are as defined above or hereinafter.
  • the substrates used, methods for producing the same, the porous polymer layers or films used, methods for producing the same, as well as methods of applying these to a surface are preferably as described hereinafter.
  • surface properties can be classified into hydrophobic and hydrophilic surfaces depending on the value of the water contact angle (WCA).
  • WCA water contact angle
  • a surface having a WCA greater than 90° is referred to as hydrophobic, whereas a WCA smaller than 90° is referred to as hydrophilic.
  • the maximum water contact angle on a smooth surface is about 120°.
  • two types of WCA values are used: static and dynamic. Static water contact angles (qstat) are obtained by sessile drop measurements, where a drop is deposited on the surface and the value is obtained by a goniometer or a specialized software.
  • Dynamic contact angles are non-equilibrium contact angles and are measured during the growth (advancing WCA qadv) and shrinkage (receding WCA qrec) of a water droplet.
  • the difference between qadv and qrec is defined as contact angle hysteresis (CAH).
  • CAH contact angle hysteresis
  • surfaces with high WCA, preferably greater than 130°, more preferably greater than 140°, most preferably greater than 150°, and/or low water CAH (less than about 30°, preferably less than about 20°, more preferably less than about 10°) are called superhydrophobic. Water droplets do not stick to such surfaces and simply roll off.
  • surfaces with both static, advancing and receding WCAs less than 20°, preferably less than 10°, more preferably less than 5°, most preferably close to or of 0° are called superhydrophilic. Water droplets are rapidly absorbed by such surfaces.
  • the substrate used is a surface having hydrophilic, preferably superhydrophilic, patterns on a hydrophobic, preferably superhydrophobic, background.
  • the patterned substrate is a surface having 6, 12, 24, 96, 384, 1536 hydrophilic, preferably superhydrophilic, areas on a hydrophobic, preferably superhydrophobic, background.
  • the porous polymer layer which exhibits hydrophobic, preferably superhydrophobic, properties is comprised of a crosslinked polyvinyl monomer, wherein the polyvinyl monomer can be one or more monomers selected from the group consisting of alkylene diacrylates, oligoethyleneoxide diacrylates or dimethacrylates, alkylene dimethacrylates, pentaerythritol tetraacrylate, pentaerythritol tetramethacrylate, trimethylopropane acrylate, trimethylopropane methacrylate, divinylbenzene, and divinylnaphthalene.
  • the polyvinyl monomer is selected from the group consisting of ethylene dimethacrylate and divinylbenzene, whereof ethylene dimethacrylate is particularly preferred.
  • the hydrophobic, preferably superhydrophobic, porous polymer may further comprise a monovinyl monomer, wherein the monovinyl monomer can be selected from the group consisting of alkyl acrylates, alkyl methacrylates, aryl acrylates, aryl methacrylates, aryl alkyl acrylates, aryl alkyl methacrylates, fluorinated alkyl acrylates, fluorinated alkyl methacrylates, styrene, vinylnaphthalene, vinylanthracene, and derivatives thereof, wherein the alkyl group in each of the alkyl monomers has 1-18 carbon atoms.
  • the monovinyl monomer is selected from the group consisting of methyl methacrylate, butyl methacrylate, benzyl methacrylate and styrene.
  • the above polyvinyl monomer is copolymerized with the monovinyl monomer affording the porous hydrophobic, preferably superhydrophobic, polymer layer.
  • the porous polymer layer comprises at least one member selected from the group consisting of porous poly(butyl methacrylate-co-ethylene dimethacrylate) (BMAcoEDMA), porous poly(methyl methacrylate-co-ethylene dimethacrylate) (MMAcoEDMA), porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethyl-acrylate) (HEMAcoEDMA), porous poly(styrene-co-1,4-divinylbenzene) (STcoDVB), porous poly(2,2,3,3,3-pentafluoropropyl methacrylate-co-ethylene dimethacrylate) (PFPMAcoEDMA).
  • BMAcoEDMA porous poly(butyl methacrylate-co-ethylene dimethacrylate)
  • MMAcoEDMA porous poly(methyl methacrylate-co-ethylene dimethacrylate)
  • HEMAcoEDMA porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethyl-
  • the pattern is created by modifying the surface of the porous polymer layer using photografting through a photomask.
  • the hydrophilic areas are created by photografting the porous polymer layer being a porous hydrophobic layer.
  • the substrate is a microarray which preferably exhibits an array-pattern which is created by photografting the porous polymer layer using a photomask.
  • photochemical methods allows precisely controlling the geometry, size and distance between the spots by the design of the photomask.
  • the porous polymer layer itself is hydrophobic, preferably superhydrophobic.
  • the hydrophobic, preferably superhydrophobic, areas originate from the porous polymer layer, whereas the hydrophilic, preferably superhydrophilic, spots/areas are created by modifying, preferably photografting the porous polymer layer.
  • the hydrophilic, preferably superhydrophilic, areas may be created by modifying, preferably photografting the porous polymer layer being a porous hydrophobic, preferably superhydrophobic, polymer layer.
  • imparting hydrophilic, preferably superhydrophilic, properties to the hydrophobic, preferably superhydrophobic, porous polymer layer is accomplished using a hydrophilic compound, for example mono- or polyvinyl monomer selected from the group consisting of monovinyl monomer, wherein the monovinyl monomer is selected from the group consisting of vinylacetate, vinylpyrrolidone, acrylic acid, methacrylic acid, methacrylamide, acrylamide, alkyl derivatives of methacrylamide, alkyl derivatives of acrylamide, wherein the alkylene group consists of 1-4 carbon atoms, hydroxyalkyl acrylates and acrylamides, hydroxyalkyl methacrylates and methacrylamides, oligoethylene glyco
  • a hydrophilic compound for example mono- or
  • said hydrophilic compound is a photografting mixture comprising [2-(methacryloyloxy)ethyl]-trimethylammonium chloride (META) or 2-acryloamido-2-methyl-1-propanesulfonic acid.
  • the hydrophilic photografting mixture consists of 15wt.% META, and 0.25wt.% benzophenone dissolved in a 1:3 (v/v) mixture of water and tert-butanol.
  • the hydrophilic areas of the substrate are separated by the hydrophobic areas so as to form an array of hydrophilic areas surrounded by a hydrophobic porous polymer surface.
  • the hydrophilic areas are separated by the hydrophobic, preferably superhydrophobic, areas of porous polymer surface so as to form hydrophilic, preferably superhydrophilic, channels.
  • the substrate comprises hydrophilic, preferably superhydrophilic, channels on a hydrophobic, preferably superhydrophobic, background and the substrate is used for microfluidic applications.
  • the hydrophilic, preferably superhydrophilic, areas which are separated by the hydrophobic, preferably superhydrophobic, areas in the substrate have a square shape.
  • Hydrophilic, preferably superhydrophilic, areas having a square shape allow dense packing of the areas and ease the readout.
  • the substrate according to the present invention is not limited to any array-format, but represents a more general and novel approach that allows patterning and for example culturing of cells in a predesigned spatial order.
  • the area-density of the hydrophilic, preferably superhydrophilic, areas is increased.
  • the amount of hydrophilic, preferably superhydrophilic, areas is at least 200 per cm 2 of the patterned substrate, more preferably at least 300 per cm 2 of the patterned substrate, even more preferably at least 400 per cm 2 of the patterned substrate.
  • the amount of hydrophilic, preferably superhydrophilic, areas is at least 500 per cm 2 of the patterned substrate.
  • the substrate used in the present invention has a size which fits on a standard microtiter scaled plate (12 x 8 cm), preferably a size of about 11 x 7 cm with at least 40000, preferably at least 50000 hydrophilic areas separated by hydrophobic, preferably superhydrophobic, background.
  • the substrate used in the present invention is a microslide of about 1 x 7 cm.
  • each of the hydrophilic, preferably superhydrophilic, areas defined in (i) has a side length of 1000 ⁇ m or less, preferably 600 ⁇ m or less, more preferably 400 ⁇ m or less, and most preferably 335 ⁇ m or less, particularly under the provision that the hydrophilic, preferably superhydrophilic, areas have a square shape.
  • the size of the hydrophilic, preferably superhydrophilic, areas is large enough to accommodate at least 100 cells, preferably 200 cells, more preferably at least 300 cells.
  • the hydrophobic, preferably superhydrophobic, areas have a width of 500 ⁇ m or less, preferably 200 ⁇ m or less, more preferably 100 ⁇ m or less and even more preferably 60 ⁇ m or less.
  • the thickness of the porous polymer layer is not limited. Regarding transparency properties or reducing manufacturing costs, a thin porous polymer layer is preferred. Particularly preferred is a porous polymer layer having a thickness of 45 ⁇ m or less, preferably 30 ⁇ m or less, and more preferably 15 ⁇ m or less.
  • the substrate used in the present can comprise at least one biologically active agent provided in at least one hydrophilic, preferably superhydrophilic, area.
  • each hydrophilic, preferably superhydrophilic, area of the patterned substrate is provided with at least one biologically active agent.
  • the substrate used in the present invention can be manufactured by a method, comprising the steps of:
  • the solid support may be of any material known in the art which is solid, preferably at a temperature within the range of -30°C to 130°C, more preferably within the range of 0°C to 40°C, more preferably at room temperature.
  • the solid support can be selected from the group consisting of glass, metal, such as a stainless steel plate, or an aluminum foil, plastic, concrete, wood, and masonry.
  • the solid support is transparent, wherein a glass solid support is particularly preferred. Such a glass solid support may be activated and/or modified prior to use.
  • the glass surface is preferably first activated, and then functionalized with an anchor-group.
  • cleaned glass plates may be immersed in 1 M NaOH for one hour and afterwards washed with deionized water followed by immersing them in 1 M HCl for 30 min, washing with deionized water and drying with a nitrogen gun.
  • the glass solid supports may be made inert for example by fluorination using for example tridecafluor-(1,1,2,2)-tetrahydrooctyltrichlorosilane placed in a vacuumed desiccator together with the glass solid support for 2h.
  • a polymerization mixture is provided between two solid supports.
  • the solid supports may be made of the same material, or of different materials.
  • the aforementioned materials suitable for the substrate according to the present invention are preferred.
  • the solid supports are made of a glass material, of which one or both may be modified and/or activated prior to use, such as a glass modified with anchor groups.
  • the solid support is a solid support as defined herein.
  • step (b) of the method for the manufacture of a substrate polymerization is carried out in the mold between the two solid supports.
  • the polymerization may involve a free radical polymerization of a polyvinyl monomer and a monovinyl monomer in the presence of porogenic solvents.
  • the free radical initiation is preferably done either thermally or by irradiation with UV- or visible light or ⁇ -irradiation.
  • the thermal initiator is generally a peroxide, a hydroperoxide, or an azo-compound selected from the group consisting of benzoyl peroxide, potassium peroxodisulfate, ammonium peroxodisulfate, t-butyl hydroperoxide, 2,2'-azobisiobutyronitrile (AIBN), and azobisisocyanobutyric acid, and the thermally induced polymerization is performed by heating the polymerization mixture to temperatures between 30°C and 120°C.
  • Polymerization can also be achieved using photoinitiators including, but not limited to, benzophenone, 2,2-dimethoxy-2-phenylaceto-phenone, dimethoxyacetophenone, xanthone, thio-xanthone, camphorquinone their derivatives, and mixtures thereof.
  • photoinitiators including, but not limited to, benzophenone, 2,2-dimethoxy-2-phenylaceto-phenone, dimethoxyacetophenone, xanthone, thio-xanthone, camphorquinone their derivatives, and mixtures thereof.
  • the polymerizing mixture is not limited, as long as said polymerizing mixture affords a porous polymer layer having hydrophobic, preferably superhydrophobic, properties. Depending on the aimed surface property of the porous polymer, i.e. hydrophobicity, the respective monomers are selected accordingly.
  • the porogen i.e. liquids that cause formation of the porous structure of the polymer
  • suitable liquid porogens include aliphatic hydro-carbons, aromatic hydrocarbons, esters, amides, alcohols, ketones, ethers, solutions of soluble polymers, and mixtures thereof.
  • water may also be used.
  • the porogen is generally present in the polymerization mixture in an amount of from about 40 to 90 vol%, more preferably from about 50 to 80 vol%.
  • the porogen is a mixture of 1-decanol and cyclohexanol.
  • the polyvinyl crosslinker is selected from the group consisting of alkylene diacrylates, alkylene dimethacrylates, alkylene diacrylamides, alkylene dimethacrylamides, hydroxyalkylene diacrylates, hydroxyalkylene dimethacrylates, wherein the alkylene group consists of 1 - 4 carbon atoms, oligoethylene glycol diacrylates, vinyl esthers of polycarboxylic acids, pentaerythritol diacrylate, pentaerythritol triacrylate, pentaerythritol dimethacrylate, and pentaerythritol trimethacrylate.
  • the monovinyl monomers are selected from the group consisting of alkyl vinylacetate, vinylpyrrolidone, acrylic acid, methacrylic acid, methacrylamide, acrylamide, alkyl derivatives of methacrylamide, alkyl derivatives of acrylamide, wherein the alkylene group consists of 1 - 4 carbon atoms, hydroxyalkyl acrylates and acrylamides, hydroxymethacrylates and methacrylamides, oligoethylene glycol acrylates and oligoethylene glycol methacrylates, potassium 3-sulfopropyl acrylate, potassium 3-sulfopropyl methacrylate, 2-acryloamido-2-methyl-1-propanesulfonic acid, 2-acrylamidoglycolic acid, [2-(methacrylooxy)ethyl] trimethylammonium chloride, and N-[3-(dimethylamino)propyl]methacrylamide.
  • alkylene group consists of 1 - 4 carbon atoms,
  • the inert porogen is selected from the group consisting of aliphatic hydrocarbons, aromatic hydrocarbons, esters, amides, alcohols, ketones, ethers, solutions of soluble polymer, water, and mixtures thereof.
  • the porous polymer layer comprises at least one member selected from the group consisting of porous poly(butyl methacrylate-co-ethylene dimethacrylate) (BMAcoEDMA), porous poly(methyl methacrylate-co-ethylene dimethacrylate) (MMAcoEDMA), porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethyl-acrylate) (HEMAcoEDMA), porous poly(styrene-co-1,4-divinylbenzene) (STcoDVB), porous poly(2,2,3,3,3-pentafluoropropyl methacrylate-co-ethylene dimethacrylate) (PFPMAcoEDMA).
  • BMAcoEDMA porous poly(butyl methacrylate-co-ethylene dimethacrylate)
  • MMAcoEDMA porous poly(methyl methacrylate-co-ethylene dimethacrylate)
  • HEMAcoEDMA porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethyl-
  • the hydrophilic porous polymer layer is made of poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) synthesized by copolymerization of 2-hydroxyethyl methacrylate (HEMA) and ethylene dimethacrylate (EDMA) in the presence of.
  • HEMA 2-hydroxyethyl methacrylate
  • EDMA ethylene dimethacrylate
  • the porous polymer layer is made of poly(butyl methacrylate-co-ethylene dimethyl-acrylate) (BMAcoEDMA).
  • BMAcoEDMA poly(butyl methacrylate-co-ethylene dimethyl-acrylate)
  • the porous polymer layer of BMAcoEDMA is synthesized by copolymerization of butyl methacrylate (BMA) and ethylene dimethacrylate (EDMA) in the presence of porogens.
  • the polyvinyl monomer is one or more monomers selected from the group consisting of alkylene diacrylates, alkylene dimethacrylates, penta-erythritol tetraacrylate, pentaerythritol tetramethacrylate, trimethylopropane acrylate, trimethylopropane methacrylate, divinylbenzene, and divinyl-naphthalene.
  • the polyvinyl monomer is selected from the group consisting of ethylene dimethacrylate and divinylbenzene, whereof ethylene dimethacrylate is particularly preferred.
  • the hydrophobic, preferably superhydrophobic, porous polymer further comprises a monovinyl monomer, wherein the monovinyl monomer is selected from the group consisting of alkyl acrylates, alkyl methacrylates, aryl acrylates, aryl methacrylates, aryl alkyl acrylates, aryl alkyl methacrylates, fluorinated alkyl acrylates, fluorinated alkyl methacrylates, styrene, vinylnaphthalene, vinylanthracene, and derivatives thereof, wherein the alkyl group in each of the alkyl monomers has 1-18 carbon atoms.
  • the monovinyl monomer is selected from the group consisting of alkyl acrylates, alkyl methacrylates, aryl acrylates, aryl methacrylates, aryl alkyl acrylates, aryl alkyl methacrylates, fluorinated alkyl acrylates, fluorinated alky
  • the monovinyl monomer is selected from the group consisting of methyl methacrylate, butyl methacrylate, benzyl methacrylate and styrene.
  • the above polyvinyl monomer is copolymerized with the monovinyl monomer in the presence of a porogenic solvent affording the porous hydrophobic, preferably superhydrophobic, polymer layer.
  • the ratio of polyvinyl monomer to monovinyl monomer is not limited as long as the obtained porous polymer exhibits hydrophobic, preferably superhydrophobic, properties.
  • the polyvinyl monomer content is within the range of 5 to 30wt.%, preferably 10 to 25wt.%, more preferably 15 to 20wt.%, based on the total amount of the polymerization mixture.
  • the monovinyl monomer content is preferably within the range of 10 to 50wt.%, preferably 15 to 45wt.%, more preferably 20 to 40wt.%, based on the total amount of the polymerization mixture.
  • Particularly preferred polymerization mixtures include:
  • step (c) After completion of the polymerization, one of the two substrates is removed in step (c).
  • the modification of the surface of the porous layer in step (d) of the method for the manufacture of a substrate is carried out by photografting the surface of the porous polymer layer using a photomask, wherein a photografting mixture is provided on the surface of the porous polymer layer.
  • the photomask may be applied so as to create the pattern, or to create the areas.
  • the photografting mixture applied to the porous polymer layer induces hydrophilic, preferably superhydrophilic, regions on the porous polymer layer.
  • the preparation of a substrate ready for further applications such as printing biomolecules takes preferably about one hour.
  • the method for the manufacture of a patterned substrate described herein preferably further comprises the step (e) providing at least one biologically active agent in at least one hydrophilic, preferably superhydrophilic, area, more preferably in each of the hydrophilic, preferably superhydrophilic, areas.
  • the substrates used, methods for producing the same, the porous polymer layers or films used, methods for producing the same, as well as methods of applying these to a surface are as described in European patent application 10 015 106.7 and/or as described in Geyer, F. L. et al., Superhydrophobic-Superhydrophilic Micropatterning: Towards Genome-on-a-Chip Cell Microarrays, Angewandte Chemie International Edition 2011, 50, pp. 8424-8427 .
  • the present invention solves the problem of creating stable, protein- and cell-repellent surfaces, as well as surfaces preventing cell migration, by coating a porous, hydrophobic surface with a layer of a water-immiscible hydrophobic liquid.
  • This creates a hydrophobic, liquid-like surface which proteins do not readily adhere to and, thus, adherent cells cannot grow, attach or migrate on this surface.
  • the water-immiscible hydrophobic liquid layer penetrates the highly porous hydrophobic polymer layer or film and does not easily wash away and thus remains stable.
  • porous hydrophilic-hydrophobic patterned surfaces can be used to pattern the water-immiscible hydrophobic liquid on the hydrophobic regions surrounded by hydrophilic areas coated with an aqueous solution and thus selectively create cell-repellent and migration-resistant areas.
  • This invention eliminates the need for physical barriers to separate cells in different spots, such as when using microtiter plates, and instead uses a liquid barrier. It is useful for applications such as cell or bacteria microarrays, cell or bacteria patterning, anti-fouling surfaces, and antimicrobial applications.
  • porous, hydrophobic polymers has been modified with a water-immiscible hydrophobic liquid to render them oleophobic, non-biofouling, and still hydrophobic.
  • these coatings are stable and self-repairing.
  • These surface characteristics are advantageous for applications which do not want non-specific protein or cell adhesion on surfaces, such as in cell patterning, cell/protein/DNA microarrays, and biomedical surfaces.
  • This invention is different from other methods, such as surface modification with PEG, because it uses a water-immiscible hydrophobic liquid as the non-biofouling component. It is also a simple and rapid method.
  • a method to modify the chemistry and properties of a porous, hydrophobic polymer with a hydrophobic liquid to render it protein- and cell-repellent and stable for cell culture is provided.
  • porous hydrophilic-hydrophobic patterned surfaces can be used to fill only the hydrophobic regions with the hydrophobic liquid, thus selectively creating protein and cell-repellent areas. This is useful e.g. for the stringent confinement of transfection mixtures and cells to localized and addressable locations for cell microarrays.
  • the hydrophobic liquid barriers allow increased spot density, confine solutions in one spot, prevent cell migration between the neighboring spots, and reduce cell adhesion on the barriers without the use of serum-free medium.
  • respective arrays are compatible with high-throughput screening methods because of the sample transparency for inverted microscopy and the standard glass slide size which is compatible with slide holders for microscopy and microarray scanners. It is envisioned that such arrays will be a useful tool for many non-biofouling applications.
  • the glass surfaces are first activated and then functionalized with an anchor group for methacrylates.
  • Activation of glass slides Clean glass slides are immersed in 1 M NaOH for 1 h and afterwards washed with deionized water, and then immersed in 1 M HCl for 30 min, washed with deionized water, and dried with a nitrogen gun.
  • Modification of glass slides Several drops of a solution containing 20 %vol. 3-(trimethoxysilyl)propyl methacrylate in ethanol, adjusted to pH 5 with acetic acid, are dropped on an activated glass slide. The plate is covered with another activated glass slide. No bubbles should be trapped between the two slides. The solution is reapplied after 30 min. After another 30 min, the slides are washed with acetone and dried with a nitrogen gun.
  • Fluorination of glass slides An activated glass slide is placed in a vacuumed desiccator together with a vial containing several drops of tridecafluoro-(1,1,2,2)-tetrahydrooctyltrichlorosilane over night.
  • Teflon film American Durafilm Co.
  • a modified glass slide is clamped on top of it.
  • the polymerization mixture is injected in between the mold and irradiated for 15 min at 12 mW/cm 2 with a 260 nm UV light.
  • the mold is then carefully opened using a scalpel.
  • An inert, fluorinated glass slide is used as the bottom plate.
  • the inability of covalent attachment of the growing polymer to the fluorinated glass slide allows for the whole polymer to stick to the top, modified glass slide during the separation process.
  • the fluorinated glass slide can be reused several times.
  • the resulting nonporous superficial layer can be easily removed by applying and rapidly removing adhesive film (Tesa tape) immediately after separating the plates while the layer is still wetted with porogen. A homogeneous, porous surface is formed.
  • the plate is washed extensively with methanol and kept in methanol for several hours to remove unreacted monomers and porogens.
  • Polymerization mixture 2-hydroxyethyl methacrylate (24 wt %), ethylene dimethacrylate (16 wt %), 1-decanol (12 wt %), cyclohexanol (48 wt %), and 2,2-dimethoxy-2-phenylacetophenone (1 wt % with respect to monomers).
  • Photografting The polymer layer is wetted with the photografting mixture and covered with a fluorinated glass slide. A photomask is placed on top and it is irradiated for 30 min with 12 mW/cm 2 260 nm UV light. The obtained pattern is washed extensively with methanol and stored in methanol for several hours to remove excess monomer and porogen, and for sterilization before cell culture.
  • Photografting mixture 2,2,3,3,3-pentafluoropropyl methacrylate (20 wt %), ethylene dimethacrylate (1.3 wt %), 1:3 (v/v) mixture of water:tert-butanol (78 wt %), benzophenone as the initiator (0.33 wt %).
  • Krytox® GPL 103 was purchased from H. Costenoble GmbH (Germany). Tetrazolium salt 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was purchased from Polysciences, Inc. (Germany). 4',6-diamidino-2-phenylindole (DAPI) was purchased from Merck KGaA (Germany). All other chemicals were purchased from Sigma-Aldrich (Germany). The glass plates were Nexterion B glass from Schott (Germany). SEM images were obtained using the LEO 1530 Gemini scanning electron microscope (Zeiss, Germany). The samples were sputtered with a thin gold layer using a Cressington 108 auto sputter coater before SEM measurement. A UK 1115 digital camera from EHD imaging (Germany) was used to take images of the water droplet on the surface under ambient conditions. Image J software with a Dropsnake plugin was used to measure the water contact angle.
  • CTC Tetrazolium salt 5-cyano
  • Pseudomonas fluorescencs Pf-05 (ATCC BAA-477) and Pseudomonas aeruginosa environmental isolate overnight liquid cultures were grown at 30 °C and 37 °C respectively in BM2 minimal medium consisting of 62 mM potassium phosphate buffer (pH 7), 7 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 10 ⁇ M FeSO 4 and 0.4 % (w/v) glucose.
  • the overnight cultures were diluted 1:10 with BM2 minimal medium to analyze the impact of Krytox® GPL 103 on bacterial growth (toxicity test) and the biofilm formation on different surfaces.
  • Krytox® GPL 103 The impact of Krytox® GPL 103 on bacterial suspensions was analyzed in a microtiter plate.
  • the microtiter plate was incubated at 30 °C on a shaker at 80 rpm. The optical density was measured after 1, 4, 8 and 22 hours at 560 nm (Labsystems Multiskan MS, Thermo Fisher Scientific, Schrete, Germany).
  • the vital staining of bacteria is based on the intracellular enzymatic reduction of CTC to red fluorescent, water-insoluble formazan crystals.
  • a DNA specific DAPI was used for the staining of the total cell count.
  • the CTC solution was freshly prepared by adding CTC to BM2 minimal medium to a final concentration of 3.8 mM.
  • the surfaces were removed from the reactor and gently washed with sterile cell wash buffer (5 mM Magnesium acetate, 10 mM Tris, pH 8.0).
  • the different surfaces were incubated in the CTC staining solution with gently shaking in darkness at room temperature (20 to 24 °C) for 3 hours.
  • the DAPI stain was added to a final concentration of 11.4 ⁇ M and incubated for 10 minutes. The surfaces were washed again with sterile cell wash buffer.
  • the bacterial adhesion on the surfaces were analyzed by epifluorescence microscopy with 200-fold magnification using the Axioplan 2 imaging system (Carl Zeiss, Oberkochen, Germany) with the filter sets for CTC (BP 546/12, FT 580, LP 590) and DAPI (G 365, FT 395, BP 445/50). Digital images of each sample were obtained with a Zeiss AxioCam MRm camera and the AxioVision 4.6 software. The surface coverage of the respiring bacteria (CTC stained, red) of five independent images per sample was determined with the BioFlux 200 software (Version 2.3.0.2; Fluxion Biosciences/ IUL Instruments GmbH, Königswinter, Germany).
  • the superhydrophobic regions were filled with a hydrophobic liquid before cell seeding ( Figure 1 ).
  • a nanoporous polymer surface with superhydrophilic spots on a superhydrophobic background was wetted with water to form droplets in the superhydrophilic spots.
  • a thin layer of hydrophobic liquid was spread over the surface while the water droplets were still contained in the superhydrophilic squares. Thus, the hydrophobic liquid only penetrated the superhydrophobic regions.
  • the hydrophobic liquid layer was washed under a stream of running water to remove the hydrophobic liquid layer from the superhydrophilic spots.
  • the hydrophobic liquid layer that fills the nanoporous, superhydrophobic polymer does not easily wash away and thus remains stable.
  • the substrate is then immersed in medium containing cells and the water droplets in the superhydrophilic squares are replaced with medium containing cells.
  • the cells preferentially adhere and grow in the superhydrophilic spots as opposed to the hydrophobic liquid background. This method eliminates the need for physical barriers to separate aqueous solutions and cells in different spots, and instead uses a liquid barrier.
  • the hydrophobic liquid used in these experiments was the Krytox fluorinated oil developed by DuPont, a low molecular weight fluorine end-capped homopolymer of hexafluoropropylene epoxide.
  • FIG 2 shows water droplets separated by Krytox barriers for various pattern geometries.
  • the superhydrophobic barriers become transparent as soon as they are filled with the hydrophobic liquid.
  • superhydrophilic-hydrophobic liquid arrays as LiquidArrays.
  • HT1080-eGFP cells were seeded at the same density on two different patterns: a square pattern with a smaller proportion of superhydrophilic area and a hexagonal pattern with a larger proportion of superhydrophilic area.
  • the hexagonal LiquidArray was able to contain the HT1080-eGFP cells within the superhydrophilic spots better than the smaller square LiquidArray and there were fewer cells on the hydrophobic liquid barriers ( Figure 4 ).
  • HEK 293 cells were cultured on LiquidArrays for 3 days and then stained with Calcein, Hoechst 33342, and propidium iodide (PI). Calcein and PI are used to indicate live and late apoptotic cells, respectively. As expected, there was low cell toxicity on both the LiquidArrays and Cassie-Baxter air arrays ( Figure 6 ). However, cells on the hydrophobic liquid barriers are not firmly adhered so some cells may wash away during the staining procedure.
  • LiquidArrays As cell microarrays, reverse cell transfection experiments were performed. First, droplets of transfection mixtures containing pCS2+-GFP and pCS2+-mCherry plasmid DNA were pipetted in a checkerboard pattern on the LiquidArray. The droplets were allowed to dry before forming the LiquidArray as described in Figure 1 . Then, HEK 293 cells were seeded on the LiquidArrays and cultured for 48 h. The cell transfection efficiency was approximately 67% and there was no mixing of transfected cells between the neighboring superhydrophilic spots ( Figure 7 ). Therefore, LiquidArrays can be used for cell patterning as well as microarray screening applications.
  • a porous BMA-EDMA polymer surface with a thickness of ⁇ 46 ⁇ m was prepared on a glass substrate.
  • the prepared surface showed a highly porous, globular structure.
  • the porosity of the surface determined by the content of porogens in the polymerization mixture, was 50%.
  • the average globule size derived from surface SEM images was 1143 ⁇ 169 nm.
  • Krytox® GPL 103 lubricant was applied to the surface, upon which the liquid easily infiltrated the hydrophobic porous polymer film forming a thin lubricating film on top of the porous polymer.
  • the pores of the porous surface became filled with the lubricant and a thin lubricant layer was fixed on the surface after the lubricant coating.
  • the static water contact angle of the surface decreased from ⁇ 139° to ⁇ 116° after lubricant coating and the lubricant coated surface also showed a much lower water contact angle hysteresis ( ⁇ 7°). These changes in water contact angles indicated that a smooth lubricant layer was formed on top of the porous surface after coating.
  • the thickness of the lubricant layer on top of the microporous BMA-EDMA surface was about 13.1 ⁇ m estimated from a weight experiment.
  • the stability of the lubricant coated surface is critical for the application of the surfaces. Mechanical damage and contamination to the surface usually decreases the receding water contact angle and thus increases the water contact angle hysteresis. Therefore, the stability of the lubricant coated surface both in air and in aqueous condition were tested by measuring the water contact angles. The water contact angles of the porous surfaces and the lubricant coated surfaces remained almost unchanged after keeping in air for 7 days, indicating that both uncoated surface and lubricant coated surface are quite stable in air.
  • the porous surfaces and the lubricant coated surfaces were fixed horizontally in a CDC biofilm reactor and a 0.94mL/min BM2 minimal medium flow was applied (corresponding to a shear force of 0.0057 N/m 2 ) inside the chamber.
  • the water contact angles of the porous surfaces changed greatly.
  • the static water contact angle of the porous surface decreased by ⁇ 10° and the water contact angle hysteresis increased significantly. It is obvious that the sheer force generated by the BM2 minimal medium flow deteriorated the uncoated surface and therefore the water contact angle hysteresis of the uncoated surfaces increased greatly.
  • the lubricant coated surface showed surprising mechanical stability under flow condition. All the static, advancing and receding water contact angles of the lubricant coated surface changed little after 7 days keeping in BM2 minimal medium flow in the CDC biofilm reactor ( Figure 8 ).
  • the Krytox® GPL 103 toxicity test showed that lubricant itself does not have an antimicrobial effect.
  • BM2 minimal medium with up to 12.5 % Krytox the bacteria showed the same growth kinetics as in pure BM2 minimal medium. After 22 hours in all samples an OD 560 nm of 0.4 was reached.
  • the lubricant coated surface also showed excellent "self-healing" properties. Once physical damage happens on the lubricant surface, the lubricant will flow toward the damaged area by a surface energy driven capillary action and spontaneously refills the physical voids.
  • the self healing properties of the lubricant coated surface were also tested. A scratch was made on the lubricant coated surface and then the scratched surface was used as surface for the 7 day bacterial experiment. It was found that there is almost no biofilm formation in the place where the surface is scratched after the 7 days bacterial experiment ( Figure 9 ), indicating that the surface showed good "self healing" properties against physical damages like scratches.
  • the present results indicate that the surfaces can inhibit Pseudomonas aeruginosa biofilm formation in the long term. Due to the good stability and the excellent biofilm inhibition properties of the lubricant coated surface, the surfaces are promising for a number of antifouling applications.

Claims (7)

  1. Substrat comportant un support solide revêtu d'un film ou couche polymère hydrophobe poreux, dans lequel ledit film ou couche polymère est revêtu d'un liquide hydrophobe non miscible dans l'eau formant un film stable sur la surface polymère ;
    dans lequel le film ou couche polymère hydrophobe poreux comprend :
    (i) des zones hydrophiles présentant des forme et taille désirées ; et
    (ii) des zones hydrophobes présentant des forme et taille désirées ;
    dans lequel les zones hydrophiles sont occupées par une solution aqueuse et seules les zones hydrophobes sont occupées par le liquide hydrophobe non miscible dans l'eau ;
    dans lequel le liquide hydrophobe non miscible dans l'eau est sélectionné à partir du groupe constitué par des lubrifiants perfluorés, des hydrocarbures perfluorés ; et des composés organosilicones ;
    dans lequel les zones hydrophiles sont entourées par les zones hydrophobes et sont séparées suivant un motif spatial désiré ; et
    dans lequel le film ou couche polymère hydrophobe poreux comprend un monomère polyvinyle réticulé et un monomère monovinyle qui ont été copolymérisés en la présence d'un porogène inerte.
  2. Procédé de formation d'une surface répulsive de protéines, cellules et bactéries, comprenant les étapes de :
    (a) préparation d'un substrat avec un support solide revêtu d'un film ou couche polymère hydrophobe poreux ; et
    (b) application d'un liquide hydrophobe non miscible dans l'eau sur ledit film ou couche polymère hydrophobe poreux ;
    dans lequel le film ou couche polymère hydrophobe poreux comprend :
    (i) des zones hydrophiles présentant des forme et taille désirées ; et
    (ii) des zones hydrophobes présentant des forme et taille désirées ;
    dans lequel une solution aqueuse est appliquée sur ledit film ou couche polymère poreux après l'étape (a) et avant l'étape (b), de telle sorte que lesdites zones hydrophiles sont occupées par ladite solution aqueuse et seules les zones hydrophobes sont occupées par le liquide hydrophobe non miscible dans l'eau ;
    dans lequel le liquide hydrophobe non miscible dans l'eau est sélectionné à partir du groupe constitué par des lubrifiants perfluorés, des hydrocarbures perfluorés et des composés organosilicones ;
    dans lequel les zones hydrophiles sont entourées par les zones hydrophobes et sont séparées suivant un motif spatial désiré ; et
    dans lequel le film ou couche polymère hydrophobe poreux comprend un monomère polyvinyle réticulé et un monomère monovinyle qui ont été copolymérisés en la présence d'un porogène inerte.
  3. Procédé destiné à empêcher ou retarder la formation de biofilms, l'adhérence de cellules et bactéries et/ou la migration de cellules et bactéries sur une surface ou sur des parties d'une surface, comprenant les étapes de :
    (a) formation sur ladite surface ou des parties de ladite surface d'un film ou couche polymère hydrophobe poreux ; et
    (b) application d'un liquide hydrophobe non miscible dans l'eau sur ledit film ou couche polymère hydrophobe poreux ;
    dans lequel ledit film ou couche polymère hydrophobe poreux comprend :
    (i) des zones hydrophiles présentant des forme et taille désirées ; et
    (ii) des zones hydrophobes présentant des forme et taille désirées ;
    dans lequel les zones hydrophiles sont occupées par une solution aqueuse et seules les zones hydrophobes sont occupées par le liquide hydrophobe non miscible dans l'eau ;
    dans lequel le liquide hydrophobe non miscible dans l'eau est sélectionné à partir du groupe constitué par des lubrifiants perfluorés, des hydrocarbures perfluorés et des composés organosilicones ;
    dans lequel les zones hydrophiles sont entourées par les zones hydrophobes et sont séparées suivant un motif spatial désiré ; et
    dans lequel le film ou couche polymère hydrophobe poreux comprend un monomère polyvinyle réticulé et un monomère monovinyle qui ont été copolymérisés en la présence d'un porogène inerte.
  4. Utilisation d'un film ou couche polymère hydrophobe poreux qui est revêtu d'un liquide hydrophobe non miscible dans l'eau afin de former une surface répulsive de protéines, cellules et bactéries ;
    dans laquelle le film ou couche polymère hydrophobe poreux comprend :
    (i) des zones hydrophiles présentant des forme et taille désirées ; et
    (ii) des zones hydrophobes présentant des forme et taille désirées ;
    dans lequel les zones hydrophiles sont occupées par une solution aqueuse et seules les zones hydrophobes sont occupées par le liquide hydrophobe non miscible dans l'eau ;
    dans lequel le liquide hydrophobe non miscible dans l'eau est sélectionné à partir du groupe constitué par des lubrifiants perfluorés, des hydrocarbures perfluorés et des composés organosilicones ;
    dans lequel les zones hydrophiles sont entourées par les zones hydrophobes et sont séparées suivant un motif spatial désiré ; et
    dans lequel le film ou couche polymère hydrophobe poreux comprend un monomère polyvinyle réticulé et un monomère monovinyle qui ont été copolymérisés en la présence d'un porogène inerte.
  5. Substrat selon la revendication 1, dans lequel le lubrifiant perfluoré est un perfluoropolyéther.
  6. Procédé selon la revendication 2 ou 3, dans lequel le lubrifiant perfluoré est un perfluoropolyéther.
  7. Utilisation selon la revendication 4, dans laquelle le lubrifiant perfluoré est un perfluoropolyéther.
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